Compositions and method for improving wound healing

ABSTRACT

The healing of wounds is promoted by contacting the wound surfaces with a suspension of collagen and a glycosaminoglycan that is chemotactic for fibroblasts and/or endothelial cells. Typical glycosaminoglycans that exhibit the desired chemotaxis are heparin, heparan sulfate, and alginate. Two or more glycosaminoglycans can be present in the suspensions. Collagen is present in the suspension in the order of 7-10 mg/ml; while the glycosaminoglycan is present in much lower concentrations, e.g., 250-350 μg/ml. Application of the collagen/glycosaminoglycan suspension to open wounds greatly increases the rate of healing.

BACKGROUND OF THE INVENTION

Human skin is a major organ of the body. It is a complex organization ofspecialized tissue cells, vascular blood supply networks, nerves,glands, lymphatic system, etc. all of which interact to form theinterface between the internal organism and the surrounding environment.In this regard the skin forms a barrier between the body and theenvironment; with one of its principal functions being the protection ofthe body from invasion by potentially hazardous materials and organisms.The skin's integrity is therefore all important to the continuedwell-being of the individual, and any breach or rupture represents athreat that must be met by the body in order to protect its containedexistence.

Breaches or ruptures in the skin's integrity can be caused by externalphysical forces such as blows, cuts, missiles etc., that penetrate anddisrupt the skin barrier; or by degenerative internal processesoccasioned by disease, congenital anomalies or changes in biochemicalsystems which result in abscesses, or ulceration that breach the skinbarrier.

Under normal circumstances the body provides mechanisms for the repairand eventual healing of the rupture or breach to thereby restore theintegrity of the skin barrier.

The repair process for even minor breaches or ruptures takes a period oftime extending from hours and days to weeks; and in some instances, asin ulceration, the breach or rupture may persist for extended periods oftime, i.e., months or even years. At all times, whether brief orextended, the potential for invasion by pathogenic organisms or foreignsubstances continues until new tissue has been generated to fully closethe rupture or breach.

Because of the danger of infection, the customary management of woundsincludes an initial thorough cleansing of the affected area to removeany contaminants such as dirt, clothing particles, or other debris whichmay introduce pathogenic materials. Any hopelessly damaged tissues maybe debrided and antiseptic materials are applied to make the area assterile as possible. If considered necessary, sutures may be used toreduce the area of the underlying tissues and thereby limit the amountof tissue exposed to subsequent contamination.

Ordinarily sterile dressings are applied to the affected area tomaintain as clean and sterile an environment as possible. The dressingsare periodically removed to permit the application of antiseptics andfurther cleansing of undesirable exudates if present. Further dressingsare then applied until the tissue rebuilding mechanisms can begin thehealing process.

The healing process is brought about by complex biological mechanismsgenerally involving several groups of special cells and proteins.Leucocytes, such as neutrophils and macrophages, crowd the wound siteand digest foreign pathogens and debris. Such cells also send outchemical signals that marshal fibroblasts in the wound vicinity andultimately generate connective structures, principally, collagen, whichmake up a major portion of the new tissues. Endothelial cells generatenew blood capillaries that grow into the reconstructed tissue areaswhere their presence is necessary to supply nutrients to the newlygrowing tissue cells and remove catabolic products. As the newcapillaries grow, the cells on the margin of the wound simultaneouslymultiply and grow inwardly. The fibrous tissue arising from this cellgrowth eventually fills the wound cavity with a network of interlacingthreads of collagen which in due time, arrange themselves in firm bandsand form the permanent new tissue.

The surface of the wound subsequently is covered by a process ofenlargement, flattening, and multiplication of the surface, orepithelial cells at the wounds' edge. These epithelial cells spread assheets into the wound, beneath the scab. Eventually the proliferatingepithelial cell sheets emanating from the wound sides coalesce to coverand close the wound on the outer surface.

All of the above noted healing processes take considerable time. Therate of healing is influenced by the wound's freedom from infection, thegeneral health of the individual, and presence of retained foreignbodies, or the like. For healthy individuals with no complications, thecompletion of healing nonetheless can take a considerable period oftime, i.e., days to weeks. In some instances, the healing process can beimpaired by constitutional deficiences, or by disease processes, andhealing may never effectively take place.

Until such time as at least superficial healing has occurred, or ifhealing is impaired, the individual remains at risk from continued ornew infection. Therefore there is a time/rate related risk factorattendant to all wound situations. The quicker the wound can heal, thesooner the risk is removed. Thus any procedure that can influence therate of wound healing, or even favorably influence the healing ofintractible wounds, would be of great value.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to compositions and procedures thatimprove the ability of wounds to heal and/or increase the rate at whichwounds heal.

More specifically, the present invention presents compositions which,when applied to wounds, greatly enhance and promote the normal healingprocesses. Such compositions comprise suspensions of a mixture of thefibrous protein, collagen; and a polysaccharide, glycosaminoglycan.

In the process of the invention, the collagen/glycosaminoglycansuspension is applied to the wound and maintained in contact therewithfor an extended period, i.e., during the entire healing process, oruntil at least closure of the wound by new tissue has taken place.

The application of the collagen/glycosaminoglycan suspension promotesthe vascularization of the wound, attracts fibroblasts and endothelialcells by chemotaxis, and generally provides a favorable environment forthe cells that participate in the healing process.

It is therefore an object of the invention to provide compositions thatpromote the tissue healing process.

It is another object of the invention to provide a method for promotingthe healing of skin tissues.

It is yet another object of the invention to providecollagen/glycosaminoglycan compositions that promote skin tissuehealing.

It is still another object of the invention to induce improved rates ofskin tissue repair by contacting wounds with an aqueous colloidalsuspension of collagen and a glycosaminoglycan.

It is yet another object of the invention to induce the healing ofpreviously intractable wounds.

Other objects and advantages of the invention will become apparent fromthe following detailed description and the claims appended hereto.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an aqueous dispersion of collagen and aglycosaminoglycan (of a special class as hereinafter defined) for use onwounds to promote the healing thereof. Most usually the aqueous phase isa standard saline solution, i.e., an isotonic salt solution, howeverwater alone may also be used.

As noted, besides the aqueous phase, there are two essential componentsin the compositions. The first of the essential components is collagen.

Collagen is a proteinaceous material comprising the major fibrouselement of mammalian skin, bone, tendon, cartilage, blood vessels, andteeth. Its biological purpose is to hold cells together in discreteunits; and secondarily it has a directive role in developing tissues.The collagen proteins are distinctive in their physical characteristicsin that they form insoluble fibers possessing high tensile strength. Itis the fibrous nature of the collagen that serves to hold the variousbody structures and components together.

While the basic molecular structure of collagen may be modified to meetthe needs of particular tissues, all collagens are organized into acommon structure consisting of three polypeptide chains that form atriple stranded helix. These triple stranded helical units, in turn, areformed into a quarter-staggered array of linearly aligned bundles whichmake up collagen fibers. The collagen fibers are stabilized by covalentcross-links.

It has been shown that purified collagen can be utilized medically inreconstructive and cosmetic surgery for the replacement of bonystructures or gaps in bony structures, and for filling out tissues wherewrinkles have formed. In such usage, collagen is secured from mammaliansources, e.g., calves, whereby extraneous proteinaceous material isremoved by various dissolution, precipitation and filtration techniquesto leave a pure collagenous product. Unfortunately this pure naturalcollagen may induce antigenic response in the host subject. Suchresponse is generated by the end portions of the collagen fibrils whichare not helically bound. Fortunately these end portions of collagen canbe cleaved therefrom by treatment with a proteolytic enzyme, e.g.pepsin. After digestion with pepsin, the cleaved peptide ends arediscarded and only the central collagen bundles (tropocollagen) remain.These central collagen bundles have greatly reduced antigenicity andthey can be used for the purposes noted above without undue antigenicside effects.

The reduced antigenic, enzyme treated collagen is an article ofcommerce. It can be secured from the Collagen Corporation of Palo Alto,Calif. under the trademarked name "Zyderm." Such purified collagen ispreferred in the present compositions.

Although reduced antigenic collagen is preferred, non-cleaved collagenthat has been isolated from animal sources may also be used. It is onlynecessary that the collagen be prepared in a sterile condition in anaqueous suspension. Some inclusion of materials commonly associated withthe collagen, e.g., polysaccharides, can be tolerated and do notinterfere with the benefits of the wound healing compositions. Otherforms of processed collagen are also useful in the compositions.

The second essential component in the present composition is aglycosaminoglycan. Glycosaminoglycans are polysaccharide materials that,in animals, form the principal component of proteoglycans found inconnective tissues. Proteoglycans consist principally of theglycosaminoglycans with minor amounts of protein. Similar molecularstructures are also found in plants.

The glycosaminoglycans, being polysaccharides, comprise repeating unitsof an amino disaccharide. The amino sugar may be a derivative of thealdose sugars, e.g., glucose, galactose in the case of animals, mannosein the case of plants etc. Some common glycosaminoglycans are heparin,heparan sulfate, keratan sulfate, chondroitin sulfate, hyaluronate, inthe case of animals; and alginate, in the case of marine plants.

It should be understood however, that only certain members of theglycosaminoglycans are useful in the wound healing compositions.Specifically, those glycosaminoglycans that exhibit chemotaxis forfibroblasts or endothelial cells are useful in the present invention.Those glycosaminoglycans that exhibit no chemotaxis, or low chemotaxis,for fibroblasts or endothelial cells are not preferred for use in thepresent compositions.

Chemotaxis can be determined by several means. Tests can be run inBoyden chambers according to the techniques taught by Boyden in Journalof Experimental Medicine Vol. 115, p. 453 et. seq. (1962). The teachingsin the publication are incorporated herein by reference. In the presentinstance, fibroblasts and/or endothelial cells are placed in thesolution in one half of the Boyden e chamber, while theglycosaminoglycan of interest is dispersed at low concentrations (e.g.10⁻¹⁰ gm/ml) in the solution in the second half of the chamber. The twohalves of the chamber are separated by a semi-permeable membrane.Migration of the fibroblasts or endothelial cells to and through thesemi-permeable membrane is subsequently noted by microscopicexamination. Those glycosaminoglycans exhibiting a strong chemotacticeffect are suitable for use in the present compositions.

In Table 1 below, there is presented some data obtained from a series ofBoyden chamber tests on collagen as well as a number ofglycosaminoglycans. In the tests the compounds of interest were testedat various concentrations for their ability to attract fibroblasts. Onlythe concentration at which the compound exhibited maximum chemotaxis isshown in the Table.

                  TABLE 1                                                         ______________________________________                                                   Concentration for                                                                           Fold increase                                                   optimal chemotaxis                                                                          in chemotaxis                                        Compound   (gm/ml)       over control (1)                                     ______________________________________                                        control    --            1.0                                                  (saline)                                                                      collagen   --            1.0                                                  chondroitin                                                                              1 × 10.sup.-10                                                                        2.3                                                  sulfate                                                                       Hyaluronic 1 × 10.sup.-8                                                                         2.0                                                  acid                                                                          Alginate   1 × 10.sup.-9                                                                         8.6                                                  Heparin    1 × 10.sup.-10                                                                        3.5                                                  ______________________________________                                    

Thus, in in vitro Boyden chamber tests, heparin and especially alginateexhibit strong chemotaxis for fibroblasts and/or endothelial cells.

In another more pertinent in vivo test technique, a piece of polyvinylalcohol (PVA) sponge ("Ivalon") is implanted in the dermis of testanimals. Test compositions, i.e. collagen and a glycosaminoglycan orcontrol materials, e.g. collagen alone, are impregnated into varioussamples of the PVA sponge prior to implantation. After a period of time,e.g., seven days, the PVA sponge is retrieved from the animal,histologically sectioned and microscopically examined for invasion ofcells and the deposition of extracellular matrix in the sponge mass. Theextent of fibroblasts and endothetial cell infiltration is a measure ofthe ability of the composition to promote healing by attraction(chemotaxis) of repair cells into the area.

In one group of experiments using the in vivo test technique outlinedabove: PVA sponge alone; PVA sponges impregnated with collagen (8.75mg/ml) alone in saline; or collagen (8.75 mg/ml) in combination withvarious glycosaminoglycans (280 μg/ml) in saline were implanted intotest animals. After 7 days, the implants were removed and examinedmicroscopically to determine the percent of sponge volume that had beeninvaded by new capillaries. Table 2 below sets forth the results:

                  TABLE 2                                                         ______________________________________                                        Volume % vascularization at 7 days after implant.                             ______________________________________                                        PVA sponge alone     0.67%                                                    Sponge + collagen alone                                                                            1.20%                                                    Sponge + collagen +  1.67%                                                    hyaluronic acid                                                               Sponge + collagen +  2.64%                                                    chondroitin sulfate                                                           Sponge + collagen + alginate                                                                       5.20%                                                    Sponge + collagen + heparin                                                                        6.66%                                                    ______________________________________                                    

From the above it will be noted that compositions including heparin andalginate exhibited a marked ability to promote vascularization.Chondroitin sulfate was significantly weaker than either heparin oralginate; while collagen alone, and collagen with hyaluronic acidappeared to be only weakly active when compared to heparin or alginate.

Thus the useful compositions of the invention comprise collagen and aglycosaminoglycan that exhibits chemotaxis for fibroblasts and/orendothelial cells. It will be understood that the glycosaminoglycans canbe present in combination in the compositions. For instance, heparinwhich exhibits a very strong ability to promote vascularization may beused in admixture with alginate, which exhibits a very strong chemotaxisfor fibroblasts. Combinations of the various glycosaminoglycans may beselected to emphasize the particular chemotactic properties of thevarious glycosaminoglycans. Heparin, heparan sulfate, and alginate arethe preferred glycosaminoglycans for use in the present compositions.Hyaluronate and chondroitin sulfate are less useful in the presentcompositions.

Heparin and heparan sulfate are staple pharmaceutic products, and arecommercially obtainable from any number of pharmaceutical sources.Alginates are produced from sea-weeds and are also readily availablefrom pharmaceutical sources. They are most commonly available as thesalt form, e.g. sodium alginate. As used herein "alginate" refers tosuch salt compositions. Although the heparins and alginates are thepreferred glycosaminoglycans for use in the healing compositions, itwill be understood that other glycosaminoglycans are also useful, solong as they exhibit a good chemotactic effect vis-a-vis fibroblastsand/or endothelial cells.

The concentrations of collagen, and especially the glycosaminoglycan, inthe aqueous dispersion must be controlled in order to achieve themaximum healing effect. In addition, the relative concentrations of thecollagen and glycosaminoglycan components must be maintained withinfairly well defined limits. If the collagen concentration is too high,there is an inhibition of the migration of fibroblast cells into thewound area. The presence of fibroblasts is vital to the ultimate repairof the damaged tissues. Similarly, if the glycosaminoglycanconcentration is too high, undesirable local hemorrhaging orinflammation may occur. But if the glycosaminoglycan concentration istoo low, fibroblast and endothelial cell migration into the wound ismarkedly reduced from the migration rates noted at optimumglycosaminoglycan levels.

Thus it has been determined that collagen should be present in theaqueous dispersion at an optimal concentration of about 7-10 mg/ml.;while the glycosaminoglycan(s) should be present at an optimalconcentration of about 250-350 μg/ml. Considerable variation above orbelow the noted concentrations is permissible. Collagen concentrationscan be in the range of several milligrams/milliliters up to perhaps15-20 milligrams/milliliter. The glycosaminoglycan(s) can be in therange of perhaps 100 micrograms/milliliter up to perhaps 1000micrograms/milliliter, so long as undue inflammation does not occur.

It is desirable to hold the concentrations close to the stated amountsto produce optimum results, and to avoid inhibitory effects orinflammatory reactions. The preferred concentration for collagen is 8.75mg/ml, and 280 μg/ml for the glycosaminoglycan(s). It should beunderstood, however, that the concentrations may be varied by perhapsone-half to twice the optimal concentrations without criticallyimpairing the usefulness of the wound healing compositions. However, atconcentrations much further below or above the optimal levels, reducedeffectiveness and undesireable side-effects can come into play.

The ratio of collagen to glycosaminoglycan is also important to theoptimal effectiveness of the compositions. Compositions showing optimalbenefit have a collagen concentration some 25-35 times greater than theglycosaminoglycan concentration. Stated in another way, theglycosaminoglycan concentration should be in the vicinity of about 3 to4% of the collagen concentration for optimal effectiveness. If thecollagen concentration in the aqueous phase is increased, then theglycosaminoglycan concentration should also be increased to maintain thedesired concentration ratio. While the noted ratios are important tomaintain the optimum healing effect, ratios diverging from those statedmay be used, but the healing effect is diminished.

The compositions of the invention are prepared from commerciallyavailable dispersions of the individual components. Collagen is normallyavailable as a saline suspension at a concentration of 35 mg/ml. Thecommercial product is diluted with sterile normal saline to theconcentration levels noted above, e.g., 8.75 mg/ml. Commercialglycosaminoglycan solution is added with good mixing to the dilutedcollagen suspension to achieve the desired level, e.g, 280 μl/ml. Theresultant colloidal suspension has a milky white appearance and has aviscosity somewhat like that of fresh egg albumen. For some purposes itmay be desirable to thicken the compositions into a more viscous orsemi-solid gelled state. If such is desired, standard medicallyacceptable gelling materials, e.g. cellulose, may be included in thecompositions.

The prepared compositions should be maintained under refrigeration; butshould not be frozen or maintained at room temperature. Freezing willinterfere with suspension properties. Ambient temperatures may permitthe growth of any minute amount of contaminants. When kept underrefrigeration, the suspensions will maintain their effectiveness forextended periods, i.e. months.

Although the compositions may be used as the aqueous colloidalsuspension of collagen and glycosaminoglycan, per se, it is alsopossible to add small amounts of an antibiotic, e.g. neomycin sulfate,normally used for topical applications. Such addition of a topicalantibiotic is not necessary to promote wound healing by the presentcompositions. Such addition is solely as a matter of convenience in thegeneral management of wounds.

In any event the wound healing compositions are used as follows:

The wound is first thoroughly cleansed and decontaminated as perstandard medical practice and any necrotic tissue is debrided to leaveas clean and sterile a wound surface as possible. A quantity of thesaline-collagen-glycosaminoglycan suspension is applied liberally to allsurfaces of the wound and a gauze dressing, thoroughly moistered withthe wound healing composition, is placed over the wound. From time totime, e.g., once or twice a day, the dressing is removed and the woundsurfaces are cleaned as in standard medical practice. The wound healingcomposition is then reapplied to the wound surfaces, and the wound iscovered with new moist gauze dressings as noted above. This procedure isfollowed until new epithelial tissue completely closes the woundsurface, at which time, application of the wound healing composition isdiscontinued.

Observation of Wound Healing Effects

As noted previously, wound healing is a complex and involved processwith features including gross anatomy, microscopic processes, chemicalchanges, cell migration etc. The most direct and reliable method ofgauging the wound repair process is histological examination. Suchexamination is, however, only semiquantitive. Nonetheless, histologydoes give a summation of all the complex processes that take placeduring wound healing. Therefore it can be used to best assess thebenefits derived from any procedures used to improve or hasten healing.

Compositions of collodial suspensions in saline of collagen alone, andcollagen with various glycosaminoglycans were applied to wounds. Afterseven days, a histologic assessment of the wound was made. Allcompositions had 8.75 mg/ml of collagen. The glycosaminoglycans werepresent at concentrations of about 280 μg/ml.

The following are descriptive composites of the histologicalassessments:

1 Collagen alone in saline: few fibroblasts; very poorneovascularization; fair degree of inflammation.

2. Collagen and heparin: virtual absence of inflammation; intensefibroplasia; extremely rich network of new capillaries and venules; thewounds at seven days gave a histologic appearance equivalent tounassisted wound repair at 3-4 weeks; extremely vascularized granulationtissue.

3. Collagen and alginate: closely similar histologically tocollagen-heparin however somewhat less vascularization thencollagen-heparin.

4. Collagen and chondroitin sulfate: much less vascularization thencollagen-heparin and collagen-alginate; appreciable inflammation.

5. Collagen and hyaluronate: marked reduction in inflammation andfibroplasia; poor vascularization.

Both collagen-heparin and collagen-alginate preparations aggressivelypromoted wound healing. Collagen-chondroitin sulfate andcollagen-hyaluronate preparations had a significantly reduced woundhealing activity. Collagen alone had no significant activity.

In view of the excellent results obtained in animal studies, someclinical tests were undertaken with the collagen-heparin preparations.The following accounts relate the results of some of these trials:

1. A 42 year old female suffering from scleroderma had chronic,recurring foot ulcers. Prior therapy had consisted of prolonged (up tosix months) bed rest and closure of the ulcer with skin grafts. In thecase of a new ulcer a collagen (8.75 mg/ml) and heparin (280 μl/ml)colloidal suspension was applied daily to the ulcerated area. Wellvascularized granulation tissue began to form promptly and the ulcerhealed completely within 4 weeks without the need for bedrest.

2. An 85 year old male with peripheral vascular insufficiency developeda large and deep ulcer over the Achilles tendon area. Conventionaltherapy failed to halt the progression of the ulcer and the patient wasconsidered to be a candidate for amputation. Treatment with thecollagen-heparin composition was commenced. After 4 months of dailyapplication of the collagen-heparin suspension, the ulcerated area wascompletely filled with well vascularized granulation tissue andepithelialization had taken place.

3. A 34 year old female with sickle cell disease suffered from recurringulcers on her lower extremities. These ulcers had required bedrest andskin grafting. Upon recurrance of an ulcer, treatment with thecollagen-heparin suspension was undertaken. The ulcer healed within sixweeks.

What is claimed is:
 1. A method for promoting the healing of a surfacewound, which method comprises:applying to the wound surface, a liquidaqueous suspension of collagen fibrils in admixture with a minor amountof a glycosaminoglycan wherein said glycosaminoglycan is chemotactic forfibroblasts or endothelial cells and the collagen is not covalentlycrosslinked to the glycosaminoglycan, wherein said liquid aqueoussuspension is applied repeatedly to the wound surface during the healingto effectively promote the healing process.
 2. The method of claim 1wherein two or more glycosaminoglycans are combined in the suspension.3. The method of claim 2 wherein the combined glycosaminoglycans areheparin and alginate.
 4. The method of claim 1 wherein theglycosaminoglycan is heparin.
 5. The method of claim 1 wherein theglycosaminoglycan is heparan sulfate.
 6. The method of claim 1 whereinthe glycosaminoglycan is an alginate.
 7. The method of claim 1 whereinthe suspension is a saline suspension.
 8. The method of claim 1 whereinthe suspension is gelled.
 9. The method of claim 1 wherein the collagenis present in the suspension in concentrations in the order of about 7to 10 milligrams per milliliter.
 10. The method of claim 1 wherein theglycosaminoglycan is present in chemotactically effective amounts. 11.The method of claim 10 wherein the glycosaminoglycan is present in thesuspension in concentrations in the order of about 100 to 1000micrograms per milliliter.
 12. The method of claim 1 wherein theglycosaminoglycan is present in the suspension in a concentration ofabout 250-350 micrograms per milliliter.
 13. The method of claim 1wherein the ratio of concentration of collagen to glycosaminoglycan isabout 25-35 to
 1. 14. A method for promoting the healing of a surfacewound, which method comprises, applying to the wound surface asuspension of fibrils, itself comprised of an admixture of:(1) collagen;and glycan, wherein the glycosaminoglycan (2) a minor amount of aglycosaminoglycan, is chemotactic for fibroblasts or endothelial cellsand the collagen is not covalently crosslinked to the glycosaminoglycan,wherein the suspension of the fibrils is applied repeatedly to the woundsurface during the healing to effectively promote the healing process.15. The method of claim 14 wherein the suspension of fibrils is a liquidsuspension of fibrils.
 16. The method of claim 15 wherein the liquidsuspension of fibrils is an aqueous suspension.
 17. A method forpromoting the healing of a surface wound, which method comprises,applying to the wound surface a suspension of fibrils, itself consistingessentially of an admixture of:(1) collagen; and (2) a minor amount of aglycosaminoglyclan, wherein the glycosaminoglycan is chemotactic forfibroblasts or endothelial cells and the collagen is not covalentlycrosslinked to the glycosaminoglycan, wherein additional suspension ofparticles is applied a number of times to the wound surface during thehealing to effectively control the healing process.
 18. The method ofclaim 17 wherein the suspension of particles is a liquid suspension offibrils.
 19. The method of claim 18 wherein the liquid suspension offibrils is an aqueous suspension.
 20. A method for promoting the healingof a surface wound, which method comprises, applying to the woundsurface a suspension of fibrils, itself comprised of an admixture of:(1)a major amount of collagen, wherein the collagen is selected fromcollagen fibrils which is not covalently crosslinked or from collagenwhich is lightly covalently crosslinked; and (2) a minor amount ofglycosaminoglycan, wherein the glycosaminoglycan is chemotactic forfibroblasts or endothelial cells and the collagen is not covalentlycrosslinked to the glycosaminoglycan, wherein additional suspension offibrils is applied repeatedly to the wound surface during the healing toeffectively promote the healing process.
 21. The method of claim 20wherein the suspension of fibrils is a liquid suspension of fibrils. 22.The method of claim 21 wherein the liquid suspension of fibrils is anaqueous suspension.
 23. A method for promoting the healing of a surfacewound which method comprises:applying repeatedly to the wound surface aliquid aqueous suspension of of collagen fibrils in admixture withglycosaminoglycan wherein said glycosaminoglycan is chemotactic forfibroblasts or endothelial cells and the collagen is not covalentlycrosslinked to the glycosaminoglycan, wherein the amounts of collagenand glycosaminoglycan in the suspension are sufficient to promotehealing, wherein the amount of collagen is below that which produces aninhibition of the migration of the cells into the wound area, and theamount of glycosaminoglycan is below that which produces undesirablelocal inflammation or hemorrhaging.
 24. A method for promoting thehealing of a surface wound, which method comprises, applying repeatedlyto the wound surface a suspension of fibrils, itself comprised of anadmixture of:(1) collagen fibrils; and (2) a glycosaminoglycan, whereinthe glycosaminoglycan is chemotactic for fibroblasts or endothelialcells and the collagen is not covalently crosslinked to theglycosaminoglycan, wherein the suspension of fibrils is appliedrepeatedly to the wound surface during the healing process, wherein theamounts of collagen and glycosaminoglycan in the suspension aresufficient to promote healing, the amount of collagen is below thatwhich produces an inhibition of the migration of the cells into thewound area, and the amount glycosaminoglycan is below that whichproduces undesirable local inflammation or hemorrhaging.
 25. Acomposition for use in promoting the healing of a surface wound,comprising a liquid aqueous suspension of a major amount of fibrils ofcollagen, in admixture with a minor amount of a glycosaminoglycan thatexhibits a chemotactic effect on fibroblasts or endothelial cells,wherein the collagen is not covalently crosslinked to theglycosaminoglylcan.
 26. The composition of claim 25 wherein two or moreglycosaminoglycans are present in the suspension.
 27. The composition ofclaim 26 wherein heparin and alginate are admixed together in thesuspension.
 28. The composition of claim 25 wherein normal salinesolution is the suspending component.
 29. The composition of claim 25wherein the aqueous suspension is gelled.
 30. A composition for use inpromoting the healing of a surface wound, comprising a liquid aqueoussuspension of fibrils of collagen in admixture with a glycosaminoglycanthat exhibits a chemotactic effect on fibroblasts or endothelial cells,wherein the collagen is not covalently crosslinked to theglycosaminoglycan and the collagen is present in the suspension at aconcentration of about 7 to 10 milligrams per milliliter.
 31. Acomposition for use in promoting the healing of a surface wound,comprising a liquid aqueous suspension of fibrils of collagen, inadmixture with a glycosaminoglycan that exhibits a chemotactic effect onfibroblasts or endothelial cells, wherein the collagen is not covalentlycrosslinked to the glycosaminoglycan, and the glycosaminoglycan ispresent in the suspension at a concentration of from about 100 to about1000 micrograms per milliliter.
 32. A composition for use in promotingthe healing of a surface wound, comprising a liquid aqueous suspensionof fibrils of collagen in admixture with a glycosaminoglycan thatexhibits a chemotactic effect on fibroblasts or endothelial cells,wherein the collagen is not covalently crosslinked to theglycosaminoglycan, and the glycosaminoglycan is present in thesuspension at a concentration of about several hundred micrograms permilliliter.
 33. A composition for use in promoting the healing of asurface wound, comprising a liquid aqueous suspension of fibrils ofcollagen in admixture with a glycosaminoglycan that exhibits achemotactic effect on fibroblasts or endothelial cells, wherein thecollagen is not covalently crosslinked to the glycosaminoglycan, and theratio of the concentration of collagen to the concentration ofglycosaminoglycan is about 25-35 to 1 by weight.
 34. The composition ofclaim 33 wherein the glycosaminoglycan is heparin.
 35. The compositionof claim 33 wherein the glycosaminoglycan is heparan sulfate.
 36. Thecomposition of claim 33 wherein the glycosaminoglycan is an alginate.37. A composition comprising a major amount of collagen in fibril form,in admixture with a minor amount of heparin, and sufficient water orsaline to suspend the collagen and the heparin, wherein the collagen isnot covalently crosslinked to the heparin.
 38. The composition of claim37 wherein the composition is chemotactic for fibroblasts or endothelialcells.
 39. The composition of claim 37 wherein collagen is present atabout 7-10 milligrams per milliliter and heparin is present at about250-350 micrograms per milliliter.
 40. A composition comprising a majoramount of collagen in fibril form in admixture with a minor amount of analginate, and sufficient water or saline to suspend the collagen andalginate.
 41. The composition of claim 40 wherein the composition ischemotactic for fibroblasts or endothelial cells.
 42. The composition ofclaim 40 wherein collagen is present at about 7-10 milligrams permilliliter and the alginate is present at about 250-350 micrograms permilliliter.
 43. A composition comprising a major amount of collagen infibril form in admixture with a minor amount of a combination of heparinand alginate, and sufficient water or saline to crosslinked to theheparin and alginate wherein the collagen is not covalently suspend thecollagen, heparin and alginate.
 44. The composition of claim 43 whereinthe composition is chemotactic for fibroblasts or endothelial cells. 45.The composition of claim 32 wherein collagen is present at about 7-10milligrams per milliliter and the heparin and alginate are present in acombined total of about 250-350 micrograms per milliliter.
 46. Acomposition comprising collagen in fibril form in admixture withglycosaminoglycan, wherein the collagen is not covalently crosslinked tothe glycosaminoglyclan, wherein the amounts of collagen andglycosaminoglycan in the suspension are sufficient to promote healing,the amount of collagen is below that which produces an inhibition of themigration of the cells into the wound area, and the amount ofglycosaminoglycan is below that amount which produces undesirable localinflammation or hemorrhaging.